1we4
From Proteopedia
| Line 1: | Line 1: | ||
| - | [[Image:1we4.gif|left|200px]] | + | [[Image:1we4.gif|left|200px]] |
| - | + | ||
| - | '''Crystal Structure of Class A beta-Lactamase Toho-1 G238C mutant''' | + | {{Structure |
| + | |PDB= 1we4 |SIZE=350|CAPTION= <scene name='initialview01'>1we4</scene>, resolution 1.70Å | ||
| + | |SITE= | ||
| + | |LIGAND= <scene name='pdbligand=SO4:SULFATE ION'>SO4</scene> | ||
| + | |ACTIVITY= [http://en.wikipedia.org/wiki/Beta-lactamase Beta-lactamase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.2.6 3.5.2.6] | ||
| + | |GENE= | ||
| + | }} | ||
| + | |||
| + | '''Crystal Structure of Class A beta-Lactamase Toho-1 G238C mutant''' | ||
| + | |||
==Overview== | ==Overview== | ||
| Line 7: | Line 16: | ||
==About this Structure== | ==About this Structure== | ||
| - | 1WE4 is a [ | + | 1WE4 is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1WE4 OCA]. |
==Reference== | ==Reference== | ||
| - | An engineered disulfide bond between residues 69 and 238 in extended-spectrum beta-lactamase Toho-1 reduces its activity toward third-generation cephalosporins., Shimizu-Ibuka A, Matsuzawa H, Sakai H, Biochemistry. 2004 Dec 21;43(50):15737-45. PMID:[http:// | + | An engineered disulfide bond between residues 69 and 238 in extended-spectrum beta-lactamase Toho-1 reduces its activity toward third-generation cephalosporins., Shimizu-Ibuka A, Matsuzawa H, Sakai H, Biochemistry. 2004 Dec 21;43(50):15737-45. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/15595829 15595829] |
[[Category: Beta-lactamase]] | [[Category: Beta-lactamase]] | ||
[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
| Line 21: | Line 30: | ||
[[Category: hydrolase]] | [[Category: hydrolase]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Mar 20 14:55:47 2008'' |
Revision as of 12:55, 20 March 2008
| |||||||
| , resolution 1.70Å | |||||||
|---|---|---|---|---|---|---|---|
| Ligands: | |||||||
| Activity: | Beta-lactamase, with EC number 3.5.2.6 | ||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||
Crystal Structure of Class A beta-Lactamase Toho-1 G238C mutant
Overview
Previous crystallographic structural analysis of extended-spectrum beta-lactamase Toho-1 predicted that the high flexibility of beta-strand B3, the region that contains a conserved KTG motif and forms one wall of the substrate-binding site, could be one of the key features contributing to Toho-1 activity toward third-generation cephalosporins. To investigate whether this possible flexibility really affects the substrate profile of this enzyme, two Toho-1 mutants have been produced, G238C and G238C/G239in, in which the glycine residue at position 238 was replaced with a cysteine and an additional glycine residue was inserted. Our intent was to introduce a disulfide bond between the cysteine residues at positions 69 and 238, and thus to lock the position of beta-strand B3. The results of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) titration indicated formation of a new disulfide bridge in the G238C mutant, although disulfide bond formation was not confirmed in the G238C/G239in mutant. Kinetic analysis showed that the activity of the G238C mutant decreased drastically against third-generation cephalosporins, while its catalytic efficiency against penicillins and first-generation cephalosporins was almost identical to that of the wild-type enzyme. This result was consistent with the prediction that flexibility in beta-strand B3 was critical for activity against third-generation cephalosporins in Toho-1. Furthermore, we have determined the crystal structure of the G238C mutant enzyme to analyze the structural changes in detail. The structural model clearly shows the introduction of a new disulfide bridge and that there is no appreciable difference between the overall structures of the wild-type enzyme and the G238C mutant, although the introduced disulfide bond slightly influenced the positions of Ser237 on beta-strand B3 and Asn170 on the Omega loop. The results of our kinetic and structural analyses suggest that the flexibility of beta-strand B3, as well as the positions of Ser237 and the Omega loop, is critical for the substrate specificity expansion of Toho-1.
About this Structure
1WE4 is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
An engineered disulfide bond between residues 69 and 238 in extended-spectrum beta-lactamase Toho-1 reduces its activity toward third-generation cephalosporins., Shimizu-Ibuka A, Matsuzawa H, Sakai H, Biochemistry. 2004 Dec 21;43(50):15737-45. PMID:15595829
Page seeded by OCA on Thu Mar 20 14:55:47 2008
